Development, Validation, and Application of an UHPLC-MS/MS Methodfor Simultaneous Determination of Vancomycin, Meropenem, andSulbactam in Human Blood Plasma and Cerebrospinal Fluid

Introduction: Postoperative intracranial infections present significant therapeutic challenges in neurosurgery, with inadequate cerebrospinal fluid drainage and antimicrobial resistance constituting primary risk factors. The escalating prevalence of extensively drug-resistant pathogens necessitates combination therapy, particularly vancomycin-meropenem-sulbactam (VCM-MER-SUL) regimens for multidrug-resistant infections. Marked interpatient pharmacokinetic variability complicates dosing, prompting the development of a UHPLC-MS/MS method for precise therapeutic drug monitoring (TDM) of these agents. Methods: Chromatographic separation was achieved on a BEH C18 column (Waters, 2.1 × 50 mm, 1.7 μm particles) with 0.1% formic acid in water (A) and methanol (B) under gradient elution conditions: 95% A; 0.7 min, 70% A; 1.0 min, 5% A; 2.0 min, 5% A; 2.1 min, 95% A; 3.5 min, 95% A. The column oven and autosampler temperatures were maintained at 37°C and 15°C, respectively. The mobile phase flow rate was set at 0.4 mL/min. The ion transition was m/z 725.5 > 144.0 for VCM, m/z 383.7 > 141.1 for MER, m/z 234.1 > 124.0 for SUL, and m/z 390.0 > 114.1 for MER-D6 (internal standard). The one-step precipitation by acetonitrile was used for sample pretreatment. Results: The calibration range for VCM and SUL was established between 0.1 and 40 mg/L, whereas for MER, it was set from 0.02 to 8 mg/L. In all cases, the linearity of the calibration curves was deemed satisfactory; the method demonstrated acceptable accuracy and precision, with intra-day and inter-day bias ranging from -9.98% to 13.40%, while the corresponding imprecision remained below 12.85%. Additionally, the variability in internal standard-normalized recovery and matrix effects was controlled, showing coefficients below 12.76% and 8.77%, respectively. Stability assessments confirmed that all analytes remained within acceptable limits under the tested conditions. Discussion: Initial trials with an acetonitrile-water mobile phase yielded poor peak symmetry and inadequate resolution for all analytes. Subsequent systematic evaluation of organic modifiers (acetonitrile vs methanol) and formic acid concentrations identified a 0.1% formic acid aqueous/methanol gradient as optimal. This condition, implemented on a BEH C18 column at 37°C, delivered symmetric peaks for vancomycin, meropenem, and sulbactam within 3.5 min—markedly faster than most reported methods. A single-step acetonitrile protein precipitation doubled vancomycin recovery relative to methanol while maintaining high efficiency for the other analytes. The method’s calibration ranges, matrix effects, recoveries, and long-term stability under clinically relevant storage and handling conditions were fully validated and concordant with literature benchmarks. Conclusion: The method is fast, sensitive, accurate, and reliable, and has been verified in the study. The streamlined one-step protein precipitation method for sample preparation, coupled with rapid chromatographic separation (3.5 minutes), demonstrated suitability for clinical applications.